Project summary Recent findings in studies of the human microbiome have added a layer of complexity to research on the impact of the diet on health. While the health benefits of certain classes of diet-derived molecules are well appreciated, the molecular mechanisms underlying these benefits have been only partially elucidated. The diet provides nutrients not only to the host but also to the microorganisms that make up the gut microbiota. The commensal metagenome, which numerically exceeds the host genome by >100-fold, contributes enormous chemical diversity to nutrient metabolism. Dietary metabolites attributable to specific gut commensals, have been identified as key effector molecules of host health and disease in several studies. At the tripartite juncture of host, diet, and microbiota, we have been investigating a unique class of sphingolipids of the gut commensal Bacteroides fragilis (BfaGCs) and their host immunomodulatory functions. BfaGCs regulate the proliferation of invariant natural killer T (NKT) cells in the host's colon, which determines disease susceptibility in the NKT cell?mediated murine model of inflammatory bowel disease. This colonic NKT-cell regulation by B. fragilis occurs only when colonization takes place during the first few days of life in mice. This observation indicates that early postnatal exposure of the gut immune system to the microbiota is crucial in establishing the number of gut NKT cells throughout life. Further studies have shown that terminal branching in the glycosphingolipid structure is crucial in directing either agonism or antagonism of NKT cells functions. It is of considerable interest that dietary branched-chain amino acids (BCAAs) can dictate the lipid structure of BfaGCs. This observation suggests a novel concept in symbiotic mediator synthesis: direct incorporation of dietary factors into bacterial biochemical pathways, where they are further converted into bioactive mediators. BCAAs are essential amino acids for humans and are primarily produced by plants. Many BCAA-rich diets of plant origin are also rich in plant oligosaccharides (POs), some of which are metabolized exclusively by B. fragilis and confer this a survival advantage to this organism in the competitive environment of the gut. We hypothesize that these phytochemicals (BCAAs and POs) act synergistically to help induce colonization by B. fragilis and promote the production of NKT cell?regulatory BfaGCs that protect the host from NKT cell?mediated colitis. Using multi-pronged approaches (bacterial genetics, gnotobiotic mouse models, and high-sensitivity analytical platforms), we propose (1) to characterize critical genes in the BCAA-derived sphingolipid biosynthetic pathway in B. fragilis and investigate their colonic NKT cell modulatory functions and (2) to determine the impact of phytochemicals on immunomodulatory BfaGC production, postnatal NKT cell development, and colitis resistance in adulthood. We expect to acquire molecular-level information on how dietary factors can synergize growth of beneficial bacteria and bacterial production of symbiotic factors.